Aggregation of spectrin and PKCθ is an early hallmark of fludarabine/mitoxantrone/dexamethasone-induced apoptosis in Jurkat T and HL60 cells

It has been shown that changes in spectrin distribution in early apoptosis preceded changes in membrane asymmetry and phosphatidylserine (PS) exposure. PKC theta was associated with spectrin during these changes, suggesting a possible role of spectrin/PKC theta aggregation in regulation of early apoptotic events. Here we dissect this hypothesis using Jurkat T and HL60 cell lines as model systems. Immunofluorescent analysis of alpha II beta II spectrin arrangement in Jurkat T and HL60 cell lines revealed the redistribution of spectrin and PKC theta into a polar aggregate in early apoptosis induced by fludarabine/mitoxantrone/dexamethasone (FND). The appearance of an alpha II beta II spectrin fraction that was insoluble in a non-ionic detergent (1% Triton X-100) was observed concomitantly with spectrin aggregation. The changes were observed within 2 h after cell exposure to FND, and preceded PS exposure. The changes seem to be restricted to spectrin and not to other cytoskeletal proteins such as actin or vimentin. In studies of the mechanism of these changes, we found that (i) neither changes in apoptosis regulatory genes (e.g., Bcl-2 family proteins) nor changes in cytoskeleton-associated proteins were detected in gene expression profiling of HL60 cells after the first hour of FND treatment, (ii) caspase-3, -7, -8, and -10 had minor involvement in the early apoptotic rearrangement of spectrin/PKC theta, and (iii) spectrin aggregation was shown to be partially dependent on PKC theta activity. Our results indicate that spectrin/PKC theta aggregate formation is related to an early stage in drug-induced apoptosis and possibly may be regulated by PKC theta activity. These findings indicate that spectrin/PKC theta aggregation could be considered as a hallmark of early apoptosis and presents the potential to become a useful diagnostic tool for monitoring efficiency of chemotherapy as early as 24 h after treatment.